Herpes simplex viruses (HSVs) cause human diseases, including cold sores, eye and genital infections, Neo-natal infections and encephalitis. They establish lifelong latent infections within sensory ganglia. The HSV envelope contains 11 virus-encoded glycoprotein's of which 4 (gD, gB and the gH-gL complex) are essential for entry. Our challenge is to define the HSV fusion machinery and determine the role of the viral and cell proteins in this process. Although it is well established that gD binds to one of several different cellular receptors, the functions of the other 3 proteins is still elusive. It is generally hypothesized that gH/gL and/or gB carry out fusion of the viral envelope with host membranes, as a consequence of gD binding to receptor. We propose 3 specific aims: 1) to further characterize gD structure and its relationship to virus entry;2) to explore the steps of virus entry that occur after gD binds its cell receptor;and 3} to carry out structure-function studies of HSV gH/gL. When gD binds HVEM, it undergoes two conformational changes as revealed by crystallographic data obtained during the previous application period. Since the C-terminal portion of the ectodomain was disordered in the crystal, it is possible that other changes occur. We hypothesize that at least one of these changes plays a role in later steps of entry. In addition, the two proteins co-crystallized as monomers and we know that each is normally oligomeric. In collaboration with Dr. A. Carfi, we solved the structure of an artificially stabilized form of dimeric gD. Though its structure fits many of our previous predictions, experiments are proposed to verify the structure and to use it for future studies. In Aim 2, we will address the following question: how does the interaction between gD and its receptor trigger downstream events that lead to virus-cell fusion? It is known that HSV enters cells by at least two different mechanisms depending on the cell type. We have preliminary evidence for a third pathway that we plan to explore. In Aim 3, we will collaborate with Dr. S. Harrison to solve the structure of gH/gL as part of our long term goal to elucidate the structure of all the HSV entry glycoproteins. Other experiments will address the function of gH/gL from both serotypes to augment the structural data and complement studies in Aims 1 and 2. The knowledge gained from these studies should contribute to our understanding of HSV entry and suggest new therapeutic approaches.